PROJECT SUMMARY Endocytosis sustains synaptic transmission. The continuous retrieval of fused synaptic vesicle membrane remnants from the presynaptic plasma membrane via the coupled, compensatory events of endocytosis replenishes the synaptic vesicle pool for sustained neurotransmitter release. Defects in synaptic vesicle endocytosis underlie Epilepsy, Down?s syndrome, Alzheimer?s, Parkinson?s and Huntington?s diseases. The molecular mechanisms that accomplish rapid synaptic vesicle endocytosis at the pre-synaptic plasma membrane are poorly understood, as are the roles of the molecules involved in effecting rapid vesicle scission. The long- term goal of this proposal is to address such issues. Dynamin 1, endophilin A1 and synaptojanin 1 are three interacting protein partners essential for rapid synaptic vesicle endocytosis. However, very little is known about the coupling or coordination of their molecular mechanisms, either in space or in time, during this membrane remodeling event. Several unknown or unresolved fundamental issues essential for understanding the roles, mechanisms, and regulation of these molecules will be addressed by the experiments proposed in this application. These include 1) the cooperative mechanisms underlying the formation of a dynamin 1-endophilin A1 copolymer around the endocytic vesicle neck, and 2) the role of the polyphosphoinositol lipid phosphatase, synaptojanin 1, in the regulation of endophilin A1- and dynamin 1-membrane interactions during membrane fission. These experiments will test the central, paradigm-shifting hypothesis that dynamin 1 and synaptojanin 1 act as regulators and/or catalysts of endophilin A1-effected membrane fission. We will use a vast array of innovative fluorescence spectroscopic and microscopic approaches, in combination with various biochemical, biophysical and cellular assays, to address these issues. These include the use of: (i) environmentally sensitive fluorescence probes for measuring protein-membrane insertion, (ii) membrane-restricted quenchers for measuring protein membrane insertion-depth, (iii) Frster resonance energy transfer (FRET) probes for determining protein polymerization and copolymerization, (iv) stopped-flow kinetics to determine the rates of protein-membrane binding, -membrane insertion, and -self-assembly, and (v) fluorescence imaging on model Giant Unilamellar Vesicles (GUVs) to visualize membrane remodeling and fission. Successful outcomes of this research will provide (i) a fundamentally improved understanding of the cooperative molecular mechanisms underlying rapid synaptic vesicle scission, and (ii) a molecular foundation for the design of drugs and therapeutics that can beneficially modulate synaptic vesicle endocytosis under various disease states.